Nucleases, including zinc finger nucleases and homing endonucleases such as SceI, that are engineered to specifically bind to target sites have been shown to be useful in genome engineering. For example, zinc finger nucleases (ZFNs) are proteins comprising engineered site-specific zinc fingers fused to a nuclease domain. Such ZFNs have been successfully used for genome modification in a variety of different species. See, for example, United States Patent Publications 20030232410; 20050208489; 20050026157; 20050064474; 20060188987; 20060063231; and International Publication WO 07/014,275, the disclosures of which are incorporated by reference in their entireties for all purposes. These ZFNs can be used to create a double-strand break (DSB) in a target nucleotide sequence, which increases the frequency of homologous recombination at the targeted locus more than 1000-fold. In addition, the inaccurate repair of a site-specific DSB by non-homologous end joining (NHEJ) can also result in gene disruption. Creation of two such DSBs results in deletion of arbitrarily large regions.
Currently, ZFNs specific for particular targets are generally identified using in vitro assays used to identify engineered zinc finger proteins. See, e.g., U.S. Patent Publication No. 20050064474. However, these in vitro assays are time and labor intensive. Furthermore, although in vitro methods accurately identify ZFPs with the desired binding activity, the architecture of ZFNs and the chromatin infrastructure over the target locus in living cells may in some instances hinder the capacity of these in vitro assays to accurately predict in vivo ZFN activity.
In vivo screening assays, particularly in yeast host cells, have been used to select homing endonucleases that bind to target sites other than their cognate binding site. See, e.g., Chames et al. (2005) Nucleic Acids Res 33(20):e178; Arnould et al. (2006) J. Mol. Biol. 355:443-458; and U.S. Patent Publication Nos. 20070117128; 20060206949; 20060153826; 20060078552; and 20040002092. However, such methods have not been broadly applied to any nuclease, including zinc finger nucleases. Moreover, previously described in vivo methods do not identify biologically active nucleases from a panel of nucleases known to bind to a specific target site, nor from a panel of nucleases known to bind to a set of sites within a particular genomic region. Rather, these previously-described in vivo screening assays utilize a randomly generated library of mutant homing endonucleases to identify proteins which bind to a particular, specific target site. Thus, previously-described assays do not predict in vivo functionality from a collection of nucleases known to bind to a particular target, nor from a collection of nucleases known to bind to a set of distinct targets within a broader genomic region. Nor do these assays accurately determine which nucleases are least toxic to the host cell.
Thus, there remains a need for additional assays to identify specific nucleases, particularly high throughput in vivo assays that identify functional, specifically-targeted nucleases.